Osteoarthritis is a painful and debilitating disease of the synovial joints, affecting an estimated 21 million people in the United States. Biomechanical factors play an important role in the normal homeostatic maintenance of the cartilage extracellular matrix by chondrocytes, and under abnormal conditions, mechanical stress may be a significant factor in the initiation and progression of joint degeneration. However, the mechanisms by which chondrocytes respond to mechanical loading of the tissue are not fully understood, and the signal transduction pathways involved in the response to mechanical or osmotic environment, either physiologically or pathologically, remain to be elucidated. The transient receptor potential vanilloid 4 (TRPV4) ion channel, a non-selective cation channel with a preference for Ca++, has been found to be involved in signal transduction in response to osmotic and mechanical stimuli in mammalian cells. The goals of this study are to characterize the role of TRPV4 in the physiological response of cartilage to mechanical loading. We hypothesize that extracellular Ca++ influx into articular chondrocytes in response to osmotic or mechanical stimuli is critically dependent on TRPV4 ion channels and is significantly modulated by inflammatory, in particular proteolytic signaling. We propose that in vivo, the loss of TRPV4 function in chondrocytes, and the associated loss of osmotic and mechanical sensitivity, will result in an imbalance of cell metabolism that induces cartilage degeneration and osteoarthritis. The specific aims of this study are: 1) To determine the role of TRPV4 in chondrocytes by conducting loss-of-function studies in porcine and murine chondrocytes in vitro, and furthermore, investigation of the modulation of TRPV4 activity by activation of proteinase-activated-receptor 2 (PAR-2), interleukin 1, leptin, or prostaglandin E2;and 2) Using gene-targeted mice, to determine the effects of inducible, cartilage-specific deletion of trpv4 on chondrocyte physiology and subsequent histological, biomechanical, and metabolic changes in their cartilage. Mice will be generated by crossing trpv4lox/lox mice with col2-cre mice, which express a constitutive or inducible CRE transgene in chondrocytes. The effects of trpv4 knockout on articular damage will be determined as a function of (i) aging, and (ii) in a pathophysiologically relevant model of osteoarthritis induced by diet-induced obesity. The long-term goals of this study are to improve our understanding of the role of mechanical factors in the regulation of cartilage metabolism in normal and diseased conditions. A better understanding of these pathways will hopefully lead to the development of new pharmaceutical or biophysical interventions for the treatment of osteoarthritis. PUBLIC HEALTH RELEVANCE: Chondrocytes in articular cartilage respond to mechanical and osmotic signals arising from joint loading;however, the cellular mechanisms by which such biophysical factors are converted to intracellular signals are not well understood. Recent evidence has shown that the Transient Receptor Potential Vanilloid 4 (TRPV4) ion channel, a non-selective cation channel with a preference for Ca++, is a primary transducer of osmotic and mechanical stimuli in mammalian cells. The goals of this study are to characterize the function of TRPV4 in articular chondrocytes in vitro, and to determine its role in an in vivo model of osteoarthritis using a transgenic mouse in which the trpv4 gene is specifically and conditionally deleted from the cartilage.